Bacterial meningitis continues to be an important cause of mortality and morbidity, including neurologic sequelae, hearing impairment and developmental delay. A major contributing factor to such mortality and morbidity is our incomplete understanding of the pathogenesis of this disease. The blood-brain barrier protects the brain from any microbes circulating in the blood. Recent studies, however, have shown that meningitis-causing pathogens penetrate the blood-brain barrier, but the microbial and host factors contributing to meningitis-causing pathogens for their penetration of the blood-brain barrier remain incompletely understood. Escherichia coli (E. coli) is the most common Gram-negative bacillary organism causing meningitis. To investigate E. coli penetration of the blood-brain barrier, we have developed the in vitro blood-brain barrier model with human brain microvascular endothelial cells (HBMEC). We have shown that meningitis-causing E. coli strains penetrate the blood-brain barrier, and identified that cytotoxic necrotizing factor 1 (CNF1) contributes to E. coli penetration of the blood-brain barrier. This was shown by our demonstration that CNF1 mutant was significantly defective in invasion of HBMEC monolayer in vitro and penetration into the brain in vivo compared to the parent E. coli strain. CNF1, the paradigm of the Rho GTPase activating bacterial proteins, however, is a cytoplasmic protein, and execution of its contribution to E. coli penetration of the blood-brain barrier requires CNF1 secretion from the bacterial cytoplasm. The secretion pathway of CNF1 remains unclear and no typical signal peptide is found in the CNF1 sequence. We hypothesize that CNF1 secretion is a novel strategy utilized by meningitis-causing E. coli to penetrate the blood-brain barrier. This hypothesis will be examined in this R21 application by investigating a secretion pathway conferred by CNF1 and determining its contribution to E. coli penetration of the blood-brain barrier. The information derived from this application is likely to help in developing a novel strategy targeting CNF1 secretion in investigating the pathogenesis, prevention and therapy of E. coli meningitis.